兔出血症病毒可视化RT-LAMP检测方法的建立与应用

发布时间：2018-06-16 15:22

  本文选题：兔出血症病毒 + RT-LAMP　； 参考：《中国兽医科学》2017年06期

【摘要】：为建立一种可视化的兔出血症病毒RT-LAMP检测方法,根据兔出血症病毒(rabbit hemorrhagic disease virus,RHDV)的主要结构蛋白VP60基因的序列设计合成4条特异性引物,以重组pMD19-T-VP60质粒为模板,通过反应条件与反应体系优化,建立RHDV的环介导等温扩增(LAMP)检测方法,测定RT-LAMP对RHDV的最低检测限,并与普通RT-PCR进行比较。结果显示,该RT-LAMP的最佳反应温度为62℃,反应时间为40 min,对RHDV的最低检测限为50 copies/L,常规RT-PCR的检测限为5×10~3copies/L,表明建立的RT-LAMP的灵敏度显著高于常规RT-PCR。建立的RT-LAMP的全部反应可在1 h内完成;反应结束后加入荧光染料Et Br可肉眼判定粉色为阳性,而棕色则为阴性,且与兔巴氏杆菌、大肠杆菌、沙门菌、产气荚膜梭菌、无乳链球菌及肺炎克雷伯菌无交叉反应。上述结果表明,建立的RT-LAMP简便、快速、灵敏、特异,且不需昂贵仪器设备,适合在基层推广应用。
[Abstract]:In order to establish a visual detection method for rabbit haemorrhagic disease virus RT-lamp, four specific primers were designed and synthesized according to the sequence of the main structural protein VP60 of rabbit hemorrhagic disease virus. The recombinant plasmid pMD19-T-VP60 was used as template. Based on the optimization of reaction conditions and reaction system, a method for detection of RHDV by ring mediated isothermal amplification (Lampp) was established, and the minimum detection limit of RT-LAMP for RHDV was determined and compared with that of common RT-PCR. The results showed that the optimum reaction temperature of RT-LAMP was 62 鈩，

本文编号：2027153